RESISTANCE OF BACTERIA 205 



in the incubator, thus providing for the development of sufficiently- 

 large colonies. At each isolation material is removed from the margin of 

 a colony in such a way as to obtain the youngest organisms, that is to 

 say, those organisms belonging to the generations most distant from the 

 bacillus from which the colony originated. After 18 subcultures the 

 bacilli have lost all resistance. They undergo bacteriophagy within 

 exactly the same time and in the same manner as do baciUi of the same 

 strain which have never been in contact with the bacteriophage. Simple 

 calculation (after having determined the number of bacilli composing a 

 colony produced under the conditions of the experiment) has shown that 

 in the case of B. dysenteriae Shiga which had acquired a refractory state 

 through cultivation in the presence of bacteriophage corpuscles of maxi- 

 mum virulence and which were then cultivated in the absence of cor- 

 puscles, the loss in resistance was complete only after 400 to 500 genera- 

 tions, that is to say, after 400 or 500 divisions. 



In comparable experiments carried out with a staphylococcus complete 

 loss of resistance was obtained after 150 to 200 generations of the ultra- 

 pure culture. 



All of these experiments agree in showing that in the absence of bac- 

 teriophage corpuscles, resistance, that is to say, the immunity acquired 

 by a bacterium, is transmitted hereditarily throughout a great many 

 generations but that it diminishes gradually with successive generations. 

 After a larger or smaller number of generations, the loss of this immunity 

 is complete. The bacterium has again become as susceptible as it was 

 prior to its conflict with the bacteriophage. 



present is not sufficient, that is, 6 or 8 drops, it should be supplemented by the ad- 

 dition of a few drops of sterile bouillon. With a fine platinum wire remove from an 

 agar slant a very small quantity of the culture which is to be purified. Introduce 

 this trace of culture into the bouillon tube removing the material by rubbing the 

 culture off on the wall of the tube at the surface of the liquid. Shake the medium 

 vigorously in order to suspend the bacteria introduced. Sterilize the platinum 

 wire and dip the end into the bouillon. Carry the trace of liquid over into the 

 condensation water of the agar tube. Shake this and then incline the tube and 

 distribute the implanted condensation water over the entire surface of the agar. 

 Place the tube vertically in the incubator. After a few attempts it is possible 

 to carry out the two procedures (that of seeding the agar and then the transfer 

 to the water of condensation) in such a way that from 5 to 10 colonies on the agar 

 slant can regularly be obtained. This method of isolation, which I believe has 

 not been published, was shown me be Dr. Roux. He employed the procedure 

 for the isolation of pure strains of B. diphtheriae in connection with his well- 

 known studies leading to the discovery of diphtheria toxin. 



